Signals generated during weight bearing exercise are involved in preservation of skeletal mass. One of the biophysical factors generated during normal loading is cell strain, or deformation. We have shown that in vitro application of dynamic mechanical strain to murine marrow cultures robustly inhibits osteoclastogenesis. The target of strain is the bone stromal cell, which responds by decreasing expression of the dominant molecule controlling osteoclast formation, RANK ligand (RANKL). The signaling mechanism activated during strain of the stromal cell which attenuates RANKL expression is the subject of this work. We have evidence that at least 2 intracellular signals are activated during strain: ERK1/2 MAP-kinase (ERK1/2MAPK) and nitric oxide (NO). Inhibition of ERK1/2MAPK prevents strain inhibition of RANKL suggesting that activation of MAPK is necessary for strain effect. Further work showing that constitutive activation of ERK1/2MAPK causes increases in NO and that nitric oxide donors decrease RANKL expression suggests that increased NO may be a distal effector of both strain and ERK1/2MAPK. Thus, our hypothesis is that mechanical strain decreases osteoclast recruitment via activation of ERKl/2MAPK, with subsequent upregulation of NO, resulting in decreased RANKL expression. We will study these mechanically induced signal transduction pathways in mechanically sensitive murine stromal cells which support osteoclastogenesis in vitro. In Specific Aim 1 we will investigate the role of strain-induced activation of ERK1/2MAPK in downstream inhibition of RANKL expression. The effect on RANKL expression of adenovirus encoding dominant-negative ERK1/2MAPK, or the dominant-negative MEK1 kinase (which blocks ERK1/2MAPK activation) will be explored in control and strained stromal cells. We will then identify the upstream effectors in the MAPK pathway using adenoviral delivery of dominant-negative small GTP-binding proteins (ras, raf, rho and cdc42) to block the strain effect. In Specific Aim 2 we will investigate NO regulation of RANKL expression. We will probe whether strain increases nitric oxide synthase isoforms. We will then ascertain whether strain induction of NO requires activation of ERK1/2MAPK, as well as whether the MAPK effect on RANKL requires NO. Finally, in Specific Aim 3, we will delineate the mechanically responsive cis-elements in the 957 bp RANKL promoter driving luciferase delivered by a non-replicating retrovirus. Deletion mapping of the RANKL promoter will allow us to localize the sequences necessary for strain inhibition of transcription. Direct regulation of the RANKL promoter by MAPK and NO will be considered. With completion of these experiments we should understand the transduction pathways by which mechanical strain regulates RANKL expression and thereby osteoclast formation.